The primary goal of this research project continues to be the elucidation of the three-dimensional structures of S-Adenosylmethionine (Adomet) synthetases from a variety of sources, including mutants. This will be accomplished by determining and analyzing the crystal structures of native enzymes as well as the structures of the complexes between these enzymes and substrates (or inhibitors). Finally, the information gained will be used to understand the functions of this important enzyme. The rational design of the potential inhibitors of the enzyme will be carried out based on the three-dimensional structural information. The structure of AdoMet synthetase crystallized in the hexagonal form has been determined at 3.0A resolution in the previous grant period. The current crystallographic R-factor is 0.32; refinement is in progress. Unfortunately, the complex between the enzyme and its substrates (ATP and L-methionine) could not be made in the hexagonal form. The preliminary study suggests that the substrates can be soaked into crystals in the tetragonal form. Therefore crystal structure analysis of the tetragonal form is in this proposal. Adequate amounts of AdoMet synthetase for this research project have been and will be provided by our collaborator, Dr. G. D. Markham. Crystallization conditions to grow relatively large tetragonal bipyramidal crystals (~0.7 nm) have been characterized in the previous grant period. The crystals belong to the tetragonal system with space group P41212 or P43212 and cell dimensions a=b=121.1, c=173.2A. Two subunits of the tetrameric enzyme are in a crystallographic asymmetric unit. The crystals diffract to about 3.5A resolution using a conventional X-ray generator at room temperature. The structure is expected to be solved with a molecular replacement method. In addition to the structure determination of the tetragonal form, the structures of several mutants will be determined in this grant period. They are low and high temperature sensitive mutants (metK 501 and 502) and a mutant in which Cys 90 and Cys 240 residues are replaced with the alanine residues. Two enzymes from new sources, a monomeric enzyme of metX gene product of E. coli and the alpha-subunit of the human enzyme, will be crystallized and their preliminary X-ray studies will be carried out.